DNA Replication PDF

DNA, Chromosomes, DNA Replication and DNA Repair What is DNA?  Features of DNA Loading… ‹#›  Figure DNA 11.8 structure Chargoff’s rule...

DNA, Chromosomes, DNA Replication and DNA Repair What is DNA?  Features of DNA Loading… ‹#›  Figure DNA 11.8 structure Chargoff’s rule A pairs with T G pairs with C Keeps width consistent Complementary DNA strands 5’ – GCGGATTT – 3’ 3’ – CGCCTAAA – 5’ Antiparallel strands a) Base pairing One strand 5’ to 3’ Key Features Other stand 3’ to 5’ Two Strands of DNA form a double helix. The bases in opposite strands hydrogen-bond according to the AT/GC rule. a) Double The 2 strands are antiparallel. helix There are approximately 10 nucleotides in each strands per complete turn of the helix. © McGraw-Hill Education 11 ‹#›  Major and minor grooves Grooves are revealed in the space-filling model Loading… Major groove Proteins bind to affect gene expression Minor groove Narrower ‹#›  To fit within bacterial cell, the chromosome must be compacted ~1000-fold The looped structure compacts the chromosome about 10-fold Loop domain s Formation of loop DNA- domains binding protein s (a) Circular chromosomal (b) Looped chromosomal DNA DNA with associated proteins Brooker, Fig 12.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display  DNA supercoiling is a second important way to compact the bacterial chromosome Supercoiling within loops creates a more compact chromosome Supercoiling (b) Looped chromosomal DNA (c) Looped and supercoiled DNA E. coli has a single chromosome with a length of 1,200um. Humans have multiple chromosomes)and the length is 19,000 to 73,000um. 12-8  Molecular structure of eukaryotic chromosomes Typical eukaryotic chromosome may be hundreds of millions of base pairs long Length would be 1 meter Chromosome Discrete unit of genetic material Chromosomes composed of DNA-protein complex ‹#› EUKARYOTIC CHROMOSOME Dependent on the cell cycle In interphase chromatin is less condensed In mitosis chromosomes condense 10,000- fold and form distinct structures. Overall organization of a eukaryotic chromosome is greater than the organization of the prokaryotic chromosome.  Model First of Chromosome level of packing Structure nucleosome 2nd level ---------------------------------------------------- 30nm fiber = chromatin requires additional proteins to those in the basic particles 3rd level ------------------------------------------------------- ------ packing of fiber itself (approx. 10,000-fold in mitotic chromosomes)  Nucleosome: Nucleosome 8 histone proteins + 146 or 147 nucleotide Histone octomer + DNA = - base pairs of DNA --- nuceotides + 2H2a + 2H2b + 2H3 + 2H4 (histone octomer) Electrostatic action between the positive charge histone and the negative phosphates of the DNA are an important stabilizing force in maintaining chromatin structure Loading…  Radial loop domains and chromatin Radial loop domains Interaction between 30-nanometer fibers and nuclear matrix Each chromosome located in discrete territory ‹#› Radial loop bound to a nuclear matrix fiber Gene Matrix-attachment regions (MARs) or G G Scaffold-attachment en en Radial e regions (SARs) e loop 25,000 to Protei 200,000 bp 30- n nm fiber MA MA fiber R R MARs are anchored to the nuclear matrix, (d) Radial loop bound to a nuclear matrix fiber thus creating radial loops Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display  Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA double helix 2 nm (a) DNA double 1 Wrapping of DNA around helix 11 nm histone proteins Histone s Nucleosome 2 Formation of a 3- (b) Nucleosomes (“beads on a string”) Histone dimensional H1 zigzag structure via histone H1 and other DNA-binding proteins (c) 30-nm 30 fiber nm 300 nm 3 Anchoring of radial loop domains to the nuclear matrix (d) Radial loop domains 4 Further compaction of radial loops to form heterochromatin 700 nm (e) 5 Metaphase chromosome with Heterochromatin 2 copies of the DNA 1,400 nm (f) Metaphase chromosome 14 a: © Dr. Gopal Murti/Visuals Unlimited; b: © Ada L. Olins and Donald E. Olins/Biological Photo Service; c: Courtesy Dr. Jerome B. Rattner, Cell Biology and Anatomy, University of Calgary; d: Courtesy of Paulson, J.R. & Laemmli, U.K. James R. Paulson, U.K. Laemmli, “The structure of histonedepleted metaphase chromosomes,” Cell, 12:817–28, Copyright Elsevier 1977; e-f: © Peter Engelhardt/Department of Virology, Haartman Institute Types of Chromatin Euchromatin - DNA that is undergoing - Appx. 90% of the DNA in the cell. Heterochromatin - ------------------in comparison to euchromatin. Genes in these regions are not expressed. ATP-Dependent Nucleosome Plows Loosen Chromatin for Gene Expression 05_29_alter_nucleosme.jpg DNA Replication Basic rules of replication Semi-conservative - Synthesis always in the 5 3’ direction - Semi-discontinuous - Origin of replication provides an opening called a replication bubble that forms two replication forks. DNA replication proceeds outward from forks Access the text alternative for slide images. ‹#›  Role of DNA polymerase DNA polymerase Covalently links nucleotides Deoxynucleoside triphosphates a) Action of DNA polymerase Access the text alternative for slide images. ‹#›  Proteins Role Figurefor necessary 11.14 of deoxynucleosideDNA replication triphosphates DNA helicase Deoxynucleoside triphosphates DNA topoisomerase Single-strand binding proteins coat the DNA strands to prevent them from re-forming a double helix. Free travels slightly ahead nucleotides Binds to ofand DNA with and the replication three fork travels phosphate 5’ to 3’ groupsATP to using removes knots Breaking separate covalent bondmove to release pyrophosphate (two ofstrand and fork forward caused by the action helicase. phosphates) provides energy to connect nucleotides DNA topoisomerase This region is the replication fork. In Relives additional coiling ahead of replication fork this diagram, it is moving from right to left. Single-strand binding proteins DNA helicase travels Keep parental strands open to act as templates along one DNA strand in the 5’ to 3’ direction and separates the DNA strands. (a deoxynucleoside triphosphate) b) Chemistry of DNA replication © McGraw-Hill Education 19 ‹#› © McGraw-Hill Education 21 Features of DNA polymerase can't begin synthesis DNA polymerase --------------------------on a bare template strand Requires a primer to get started - DNA primace makes the primer from RNA The RNA primer is removed and replaced with DNA later DNA polymerase only works ‹#› Why does DNA replication only occur in the 5’ to 3’ direction? ⑯ ↓ Should be PPP here  Comparison of the leading and lagging strands Leading strand molecule - DND synthesized in as one long - DNA primace makes a single RNA primer DNA polymerase adds nucleotides in a 5’ to 3’ direction as it slides forward Lagging strand Okazaki -DNA synthesized sto 3' out as Okazaki fragments consist of RNA primers plus DNA fragments In both strands RNA primers are removed by DNA polymerase and replaced with DNA DNA ligase joins adjacent DNA fragments ‹#›  Figure 11.18 separation of know this DNA here startshisnagmentfig, as disco 3rd > - semi discontinuous 34d Enq 1st 1 O. Of rep. ‹#› Figure 11.19 Access the text alternative for slide images. ‹#›  Core proteins at the replication fork Topoisomerases - Prevents torsion by DNA breaks Helicases - - separates 2 strands Primase - RNA primer synthesis single strand- - prevent reannealing binding proteins of single strands DNA polymerase - synthesis of new strand Camp - stabilizes polymerase DNA ligase - seals nick via phosphodiester linkage camp loader ↳ Emereses  DNA replication is very accurate Three mechanisms for accuracy Hydrogen bonding between A and T, and between G and C is more stable than mismatched combinations DNA polymerase - Active Loading… site polymerase of DuA is unlikely to form : bonds if mismatched. pairs editing DNA polymerase can proofread to remove 3" -S mismatched pairs ↳ DNA polymerase backs up and digests linkages well ↳ Other DNA repair enzymes as & ‹#›  DNA Polymerases 1 E. coli has 5 DNA polymerases DNA polymerase multiple subunits, responsible for -----------------– majority of replication DNA polymerase I a single subunit, rapidly removes -----------------– RNA primers and fills in DNA DNA polymerse # , I -----------------------------------– , DNA repair and can replicate damaged DNA I DNA polymerases ------------stall at DNA damage t #, DNA polymerases ----------------don’t stall but go slower and make sure replication is complete ‹#›  DNA Polymerases 2 Humans have 12 or more DNA polymerases Designated with Greek letters ers DNA polymerase α – - its own built in primace subunit DNA polymerase δ an ε – extend DNA at a faster rate d DNA polymerase γ – replicates mitochondrial DNA polymerase When DNA polymerases α, δ ε encounters or toencounter abnormalities they may be unable replicate Lesion-replicating polymerases may be able to synthesize complementary strands to the damaged area ‹#›  Telomeres Series of short nucleotide sequences repeated at the ends chromosomes eukaryotes of in Specialized form of DNA replication only in eukaryotes in the telomeres Telomere at 3’ does not have a complementary strand and is called 3 overhang Telomerase fixes it selomerase during cancer - , ⑳ · turns on. Telomerases are usually turned off soon after your birth. ‹#›  Figure 11.20 https://www.youtube.com/watch?v=8unMOUySGYY ‹#›  Telomerase functions Shortening of telomeres is correlated with cellular senescence* finite lifetime a Telomerase function is reduced as an organism ages 99% of all types of human cancers have high levels of telomerase ‹#› DNA Repair and Recombination Mutation change heritable A ------------------in the genetic material Essential to the continuity of life Source of variation for natural selection New mutations are more likely to be DNA repair systems reverse Cancer is a disease caused by gene mutations ↓ in tumor supressor genes Causes of DNA Damage - Copying error from DNA Polymerase DNA Pol a and e have 3’-5’ exonuclease activity - chemical damage Endogenous (radicals formed as a result of metabolism) Exogenous (environmental) Ames Test - Radiation Damage Ionizing radiation causes DNA breaks U.V. radiation causes DNA distortions (T-C, C-C, T-T dimers) Types of DNA Damage Point mutations Deamination - Dupurination Depyrimination DNA distortions (T-C, T-T dimers) - Interstrand Crosslinks DNA-protein crosslinks - Strand breaks 06_23_Depurination.jpg 06_25_mutations.jpg Point mutation examples Base substitution 5’ – GGCGCTAGATC – 3’ 5’ – GGCGCTAGATC – 3’ → 3’ – CCGCGATCTAG – 5’ 3’ – CCGCGATCTAG – 5’ Add or delete a single base pair 5’ – GGCGCTAGATC – 3’ 5’ – GGCAGCTAGATC – 3’ → 3’ – CCGCGATCTAG – 5’ 3’ – CCGTCGATCTAG – 5’ Copyright © 2017 McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. ‹#› Table 14.1 know This ! Gene mutations may affect amino acid sequences - Silent mutation Does not alter the amino acid sequence Due to degeneracy of genetic code - Missense mutation Changes a single amino acid in a polypeptide May not alter function if substituted amino acid is similar in chemistry to original ex: Sickle-cell disease Gene mutations may affect amino acid -- sequences continued - - Nonsense Mutation Change from a normal codon to a stop codon Produces a truncated polypeptide - Frameshift mutation Addition or deletion of nucleotides (excluding multiples of 3) Completely different amino acid sequence downstream from mutation  Gene mutations outside of coding..... sequences A mutation may alter the sequence within a --------and promoter affect the rate of transcription May enhance or inhibit transcription elements regulatory Mutations may occur in other -------------------or operator sites Mutation may alter DNA sequence of operator so that repressor protein does not bind Table 14.2 Jump to long image description Copyright © 2017 McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. ‹#›  Germ-line or somatic cell mutations - The time a location of a mutation determines its severity and the heritability. Germ-line cells give rise to gametes Mutation can occur in sperm or egg cell, or in gamete progenitor cells Somatic cells are all other body cells Can occur early or late in development Gives a genetic mosaic with patches of mutant tissue Table 14.3 Mutagens alter DNA - Disruption of base-pairing Some modify nucleotide structure Nitrous acid deaminates bases, changing C to U, so that it pairs with the wrong nucleotide Mustard gas or EMS alkylate bases, adding methyl or ethyl groups Base analogues substitute into DNA - Disruption of replication Some insert between the bases and distort the helix Benzopyrene, found in cigarettes and charbroiled food Physical mutagens - Radiation damage radiation ionizing ---------------has high energy and penetrates deeply to create free radicals X rays and gamma rays Cause deletions or breaks in one or both DNA strands radiatio nonionizing i -------------------has less energy and can only penetrate the surface UV rays can cause formation of thymine dimers, causing gaps or incorporation of incorrect bases 06_24_radiation.jpg 06_23_Depurination.jpg DNA Repair Mechanisms Direct chemical reversal -----------------------------of the damage Excision Repair damaged base or bases are removed and then replaced with the correct ones - Base exclusion repair - Nucleotide excision repair - Mismatch repair Base Excission Repair DNA glycosylase -------------------removes the damaged base occurs about 20,000 times a day in every cell Removal of its deoxyribose phosphate produces a gap Correct nucleotide is incorporated by DNA polymerase B - Ligation  Nucleotide Excision Repair The damage is recognized by one or more protein factors that assemble at the location. The DNA is unwound producing a "bubble". - Cuts are made on both s side and s'side of the damaged area of the tract containing the damage can be removed Using the opposite strand as a template DNA Pol or E fills in the correct nucleotides This is followed by Ligation Nucleotide-excision repair proceeds most rapidly on the DNA strand that is serving as the template for transcription. Figure 14.8 Jump to long image description Copyright © 2017 McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. ‹#› Mismatch Repair (MMR) Mismatch repair deals with correcting mismatches of the normal bases - Accounts for ge % of all repairs Follows behind replication fork - Recognition of Mismatch requires protein complex Excision of mismatch - DNA synthesis by Pol of or 3. 06_13_polymerase1.jpg Missense mutations Chemical mutagens have been shown to cause missense mutations leading to cancer that W genes - Proto-onesgone · in have normal function the all. that Oncogene protooncogene - · has mutected I has been become hyperactive. Gene amplifications Increase in copy number results in too much protein Many human cancers are associated with amplification of particular proto-oncogenes Chromosomal translocations Two chromosomes break and switch ends Very specific translocations associated with certain types of tumors Can create chimeric genes Figure 14.14 Retroviral insertions Viral DNA inserts into a chromosome, putting a viral promoter next to a proto-oncogene If proto-oncogene becomes overexpressed, it will promote cancer Some viruses cause cancer because they carry an oncogene in the viral genome Repairing Strand Breaks Single-Strand Breaks uses the same enzyme systems that are used in base excision repair Double-Strand Breaks Nonhomologous End Joining = Direct joining of the broken ends - Recombination Momologous Homologous Recombination Homologous Recombination Transposable element: mobile genetic elements of a chromosome that have the capacity to move from one location to another in the genome. Normal and ubiquitous ------------------------ components of prokaryote and eukaryote genomes. Prokaryotes-transpose to/from cell’s chromosome, plasmid, or a phage chromosome. to /from same a different chromosome Eukaryotes or - - transpose Nonhomologous recombination: transposable elements insert into DNA that has no sequence homology with the transposon. Transposable elements cause genetics changes and make important contributions to the evolution of genomes: - Insert into genes Insert into regulatory sequences; modify gene expression. - Produce chromosomal mutations.  Transposable elements: Two classes of transposable elements/mechanisms of movement: 1. Encode proteins that (1) move DNA directly to a new position or (2) replicate DNA and integrate replicated DNA elsewhere in the genome (prokaryotes and eukaryotes). > mechanism 1. - cut a poste paste mechanism 2. - copya Retrotransposons encode neverse transcriptase and make DNA 2.- ----------------------------------------------------------------------------------- copies -; new DNA copies integrate at different sites (eukaryotes only). RNA transcripts of ↓ Broad Institute, MIT Transposable elements in prokaryotes: Two examples: 1. Insertion sequence (IS) elements 2. Transposons (Tn) Insertion sequence (IS) elements: 1. Simplest type of transposable element found in bacterial chromosomes and plasmids. mobilization and gene (transposense) 2. - Encode for insertion 3. Range in size from 768 bp to 5 kb. all IS elements shows IRS (inverted - Ends of known 4. replet sequences) 1. Transposition initiates when transposase recognizes ITRs. - Site of integration - target Site Staggered cuts are made in DNA at target site by transposase, IS element inserts, DNA polymerase and ligase fill the gaps (note--- transposase behaves like a restriction enzyme). - small direct repeats the target flanking created. size are Transposons (Tn): Similar to IS elements but are more complex structurally and carry additional genes 2 types of transposons: 1. - Composite transposons Non composite transposons 2. - Composite transposons (Tn): Carry genes (example might be a gene for antibiotic resistance) flanked on both sides by IS elements. Tn10 is 9.3 kb and includes 6.5 kb of central DNA (includes a gene for tetracycline resistance) and 1.4 kb inverted IS elements. IS elements supply transposase and ITR recognition signals. Fig. 7.21a Noncomposite transposons (Tn): resistance) but don't terminate - Larry genes (ex. ant. with IS elements Ends are non-IS element repeated sequences. Tn3 is 5 kb with 38-bp ITRs and includes 3 genes; bla ( -lactamase), tnpA (transposase), and tnpB (resolvase, which functions in recombination). Fig. 7.21b Human retrotransposons: Alu1 SINEs (short-interspersed sequences) X 000-500 , 000 ~- 300 up long , repeated 300 , Flanked by 7-20 bp direct repeats. RNA intermediate - some are transcribed , thought to more by AluI SINEs detected in neurofibromatosis (OMIM1622200) intron; results in loss of an exon and non-functional protein. L-1 LINEs (long-interspersed sequences) 6.5 kb element, repeated 50,000-100,000X (~5% of genome). - Contain ORFs with homology to reverse lacks LTRs transcriptases ; Some cases of hemophilia (OMIM-306700) known to result from newly transposed L1 insertions.  Models of transposition: Similar to that of IS elements; duplication at target sites occurs. Transposition may be replicative (duplication = copy and paste), but it can also be non-replicative (transposon lost from original site = cut and paste). Movement of a transposon from one genome (e.g., plasmid) to another (e.g., chromosome)can cointegrate transposon to both genomes (duplication) in the case of replicative. Result in same types of mutations as IS elements: insertions, deletions, changes in gene expression, or duplication.